This proposal continues our investigations of the mechanism of transcriptional activation by the bovine papillomavirus (BPV) E2 protein. E2 is a sequence specific DNA binding protein that coordinates viral transcription. Together with the E1 protein which it binds, BPV E2 is necessary for viral DNA replication in vivo, although the properties of E2 necessary for stimulating replication have not been determined. Our investigations of E2 include comprehensive mutational and biochemical analyses of the domains involved in transcriptional activation and DNA binding. To understand the mechanisms by which E2 activates transcription, we previously proposed experiments to identify cellular proteins that interact with the E2 transcriptional activation domain, as currently none are known. During the current funding period two novel cDNAs were cloned using the yeast two hybrid system. These proteins have been shown to bind specific regions of E2 in vitro. Genetic experiments with one clone suggest its interaction with E2 is necessary for both E2 transcription and replication activities. The characterization of these two cellular proteins and the significance of their interaction with E2 are major goals of this renewal. We have also found that E2 binds several known cellular transcription factors, and correlations with mutational data will be used to determine their functional significance. We have previously reported studies of the C-terminal E2 DNA binding domain and the amino acids involved in nucleic acid recognition and dimerization. Our genetic analyses indicated that this region of E2 also plays an important role in transcriptional activation. In collaboration with Dr. James Baleja we have applied high resolution NMR to study the structure of the E2 C-terminus, enabling detailed correlations with our mutational analyses of E2. These structural techniques will now be applied to examine the protein interaction domains in E2 and can be used to determine the molecular conformation of the E2 transactivation domain. This information, together with our identification and characterization of cellular proteins and transcription factors that assemble on E2, should provide novel insights into the mechanism of action of this important protein.